Omega-3 PUFA ameliorated serotonin-related bone loss in response to early life stress followed by subsequent chronic mild stress in ovariectomized rats

Abstract

Maternal separation (MS) at early life induces long-term serotonin dysfunction, resulting in increased susceptibility to chronic mild stress (CMS) in adulthood. Omega-3 polyunsaturated fatty acid (PUFA) is known to promote stress resilience by interacting with central serotonergic systems, but it remains unknown if omega-3 PUFA plays bone-protective role through hypothalamic serotonin 2C receptor (5HT2CR). The present study was to investigate preventive effects of omega-3 PUFA on stress-induced bone loss by altering 5HT2CR signaling, conferring greater resilience to two-hit stress (CMS+MS) compared to CMS alone. Female pups were fed a modified AIN-93G diet with 0% or 1% omega-3 PUFA relative to total energy intake during the pre-weaning (embryonic day (ED) 0−postnatal day (PND) 20), post-weaning (PND 21−PND 108), or lifetime period (ED 0−PND 108). The rats were further divided into three conditions of stress, non-stress, CMS, and two-hit stress. MS was conducted in early life (PND 2−14) and CMS was conducted in adulthood (PND 91−105), 1 week after ovariectomy or sham surgery. Regardless of omega-3 PUFA supplementation, CMS and two-hit stress reduced serotonin levels in brainstem with greater reduction in two-hit stress groups. The corresponding down-regulated hypothalamic expressions of 5HT2CR, calmodulin kinase IV (CaMKIV), phosphorylated CaMKIV (pCaMKIV), cAMP response element binding protein (CREB), and phosphorylated CREB (pCREB) were observed. The increased malondialdehyde (MDA) levels in bone and deterioration of trabecular microstructure including femoral bone volume (BV), BV/tissue volume and porosity were detected. Omega-3 PUFA supplementation during lifetime increased brainstem serotonin levels, hypothalamic expressions of 5HT2CR, and its downstream signaling mediators compared to control diet. The following activation of 5HT2CR pathway decreased MDA levels in bone and improved trabecular microstructure. Neither pre-weaning nor post-weaning supplementation of omega-3 PUFA led to a significant upregulation of 5HT2CR signaling and improvement of bone structure. In conclusion, the present study suggested that lifelong omega-3 PUFA supplementation ameliorated stress-induced bone loss via 5HT2CR in ovariectomized rats, which its resilience to CMS and two-hit stress was comparable.